The present invention relates generally to the detection and isolation of fumonisin resistant organisms and to compositions and methods for the in vivo detoxification or degradation of fumonisin. This method has broad application in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.
Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants. These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop losses, is interference with the mechanism by which the pathogen causes injury to the host crop plant. Still another method, in the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, is interference with toxin production, storage, or activity. This invention falls into the latter two categories.
Since their discovery and structural elucidation in 1988 (Bezuidenhout S, Gelderblom W, Gorst-Allman C, Horak R, Marasas W, Spiteller B, Vleggaar R (1988) xe2x80x9cStructure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme.xe2x80x9d Journal Chem Soc, Chem Commun 1988: 743-745), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are linked to several animal toxicoses including leukoencephalomalacia (Marasas W F O, Kellerman T S, Gelderblom W C A, Coetzer J A W, Thiel P (1988) xe2x80x9cLeukoencephalomalacia in a horse induced by fumonisin B-1 isolated from Fusarium moniliforme.xe2x80x9d Onderstepoort Journal of Veterinary Research 55: 197-204; Wilson T M, Ledet A E, Owens D L, Rice L G, Nelson H A (1990) xe2x80x9cExperimental liver disease in ponies associated with the ingestion of a corn-based ration naturally contaminated with fumonisin B1,xe2x80x9d American Association of Veterinary Laboratory Diagnosticians: Abstracts 33rd Annual Meeting, Denver, Colo., Oct. 7-9, 1990., Madison, Wis., USA) and porcine pulmonary edema (Colvin B M, Harrison L R (1992) xe2x80x9cFumonisin-Induced Pulmonary Edema and Hydrothorax in Swine.xe2x80x9d Mycopathologia 117: 79-82). Fumonisins are also suspected carcinogens. (Geary W (1971) Coord Chem Rev 7: 81; Gelderblom W C A, Kriek N P J, Marasas W F O, Thiel P G (1991) xe2x80x9cToxicity and Carcinogenicity of the Fusarium-Moniliforme Metabolite, Fumonisin-B1, in Rats.xe2x80x9d Carcinogenesis 12: 1247-1251; Gelderblom W C A, Semple E, Marasas W F O, Farber E (1992) xe2x80x9cThe Cancer-Initiating Potential of the Fumonisin-B Mycotoxins.xe2x80x9d Carcinogenesis 13: 433-437). Fusarium isolates in section Liseola produce fumonisins in culture at levels from 2 to  greater than 4000 ppm (Leslie J, Plattner R, Desjardins A, Klittich C (1992) xe2x80x9cFumonisin B1 production by strains from different mating populations of Gibberella fujikoroi (Fusarium section Liseola).xe2x80x9d Phytopathology 82: 341-345). Isolates from maize (predominantly mating population A ) are among the highest producers of fumonisin. (Leslie et al., supra). Fumonisin levels detected in field-grown maize have fluctuated widely depending on location and growing season, but both preharvest and postharvest surveys of field maize have indicated that the potential for high levels of fumonisins exists (Murphy P A, Rice L G, Ross P F (1993) xe2x80x9cFumonisin-B1, Fumonisin-B2, and Fumonisin-B3 content of Iowa, Wis., and Illinois corn and corn screenings.xe2x80x9d J Agr Food Chem 41: 263-266). Surveys of food and feed products have also detected fumonisin (Holcomb M, Thompson H C Jr., Hankins L J (1993) xe2x80x9cAnalysis of fumonisin B-1 in rodent feed by gradient elution HPLC using precolumn derivation with FMOC and fluorescence detection.xe2x80x9d J Agr Food Chem 41: 764-767; Hopmans E C, Murphy P A (1993) xe2x80x9cDetection of Fumonisin-B(1), Fumonisin-B(2), and Fumonisin-B(3) and hydrolyzed Fumonisin-B(1) in Coin-Containing foods.xe2x80x9d J Agr Food Chem 41: 1655-1658; Sydenham E W, Shephard G S, Thiel P G, Marasas W F O, Stockenstrom S (1991) xe2x80x9cFumonisin Contamination of Commercial Corn-Based Human Foodstuffs.xe2x80x9d J Agr Food Chem 39: 2014-2018). The etiology of Fusarium ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson P E (1992) xe2x80x9cTaxonomy and Biology of Fusarium moniliforme.xe2x80x9d Mycopathologia 117: 29-36). Fusarium can be isolated from most field grown maize, even when no visible mold is present. The relationship between seedling infection and stalk and ear diseases caused by Fusarium is not clear. Genetic resistance to visible kernel mold has been identified (Gendloff E, Rossman E, Casale W, Isleib T, Hart P (1986) xe2x80x9cComponents of resistance to Fusarium ear rot in field corn.xe2x80x9d Phytopathology 76: 684-688; Holley R N, Hamilton P B, Goodman M M (1989) xe2x80x9cEvaluation of tropical maize germplasm for resistance to kernel colonization by Fusarium moniliforme.xe2x80x9d Plant Dis 73: 578-580), but the relationship to visible mold to fumonisin production has yet to be elucidated.
Fumonisins have been shown in in vitro mammalian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphinganine acyl transferase, resulting in the accumulation of the precursor sphinganine. (Norred W P, Wang E, Yoo H, Riley R T, Merrill A H (1992) xe2x80x9cIn vitro toxicology of fumonisins and the mechanistic implications.xe2x80x9d Mycopathologia 117: 73-78; Wang E, Norred W, Bacon C, Riley R, Merrill A Jr. (1991) xe2x80x9cInhibition of sphingolipid biosynthesis by fumonisins: implications for diseases associated with Fusarium moniliforme.xe2x80x9d J Biol Chem 266: 14486; Yoo H S, Norred W P, Wang E, Merrill A H, Riley R T (1992) xe2x80x9cFumonisin Inhibition of de Novo Sphingolipid Biosynthesis and Cytotoxicity Are Correlated in LLC-PK1 Cells.xe2x80x9d Toxicol Appl Pharmacol 114: 9-15) It is likely that inhibition of this pathway accounts for at least some of fumonisin""s toxicity, and support for this comes from measures of sphinganine:sphingosine ratios in animals fed purified fumonisin (Wang E, Ross P F, Wilson T M, Riley R T, Merrill A H (1992) xe2x80x9cIncreases in Serum Sphingosine and Sphinganine and Decreases in Complex Sphingolipids in Ponies Given Feed Containing Fumonisins, Mycotoxins Produced by Fusarium moniliforme. xe2x80x9cJ Nutr 122: 1706-1716). Fumonisins also affect plant cell growth (Abbas H K, Boyette C D (1992) xe2x80x9cPhytotoxicity of fumonisin B1 on weed and crop species.xe2x80x9d Weed Technol 6: 548-552; Vanasch M A J, Rijkenberg F H J, Coutinho T A (1992) xe2x80x9cPhytotoxicity of fumonisin B1, moniliformin, and t-2 toxin to corn callus cultures.xe2x80x9d Phytopathology 82: 1330-1332; Vesonder R F, Peterson R E, Labeda D, Abbas H K (1992) xe2x80x9cComparative phytotoxicity of the fumonisins, AAL-Toxin and yeast sphingolipids in Lemna minor L. (Duckweed).xe2x80x9d Arch Environ Contam Toxicol 23: 464-467). Kuti et al. xe2x80x9cEffect of fumonisin B1 on virulence of Fusarium species isolated from tomato plants.xe2x80x9d (Abstract, Annual Meeting American Phytopathological Society, Memphis, Tenn.: APS Press 1993) reported on the ability of exogenously added fumonisins to accelerate disease development and increase sporulation of Fusarium moniliforme and F. oxysporum on tomato.
The toxicity of fumonisins and their potential widespread occurrence in food and feed makes it imperative to find detoxification or elimination strategies to remove the compound from the food chain.
The present invention provides newly discovered enzymes capable of degrading and detoxifying fumonisins, produced by fermentation of one or more of Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552. The invention further comprises methods for making enzymes that are capable of detoxifying fumonisins, comprising the step of growing one or more of Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium ATCC 55552 in the presence of a fumonisin or the metabolite produced by action of the enzyme on a fumonisin. Alternatively, enzymes are isolated from the seeds or plant parts of a plant transformed and expressing a fumonisin esterase. This invention further provides methods of detoxifying fumonisins, comprising the step of reacting fumonisin with an enzyme derived from Exophiala spinifera, ATCC 74269, Rhinocladiella atrovirens, ATCC 74270, or the bacterium of ATCC 55552. Fumonisin can be degraded in harvested grain, during the processing of harvested grain, in animal feed, or in plant tissue as, for example, during the use of the plant for silage or as a spray on grain, fruit or vegetables. In addition, the invention provides a method of detoxifying a fumonisin, a structurally related mycotoxin, a fumonisin hydrolysis product, or a hydrolysis product of a structurally related mycotoxin, comprising reacting the said toxin with an AP1 catabolase.
Genes that code for the fumonisin-degrading enzyme for Exophiala spinifera, ATCC 74269 (ESP1) and the bacterium of ATCC 55552 (BEST) have been isolated and sequenced and the amino acid and DNA sequence of the enzymes are provided here. It is known that genes encoding proteins, such as the fumonisin-degrading enzymes, can be identified, isolated, cloned and expressed in transgenic organisms, including several important crop plants. In addition two short amino acid domains of ATLM and TNI are unique to fumonisin esterase and are not found in other known esterase.
This invention also provides a mechanism for selection of transformants: growth of plant cells in the presence of a Fusarium or its mycotoxin favors the survival of plant cells that have been transformed to express the coding sequence that codes for the enzyme of this invention and degrade the toxin. Alternatively, a phytohormone is linked to a tricarballylic acid (TCA) rendering the phytohormone inactive until cleaved by an esterase. When the inactive phytohormone is added to the culture medium only plants expressing an esterase will be able to grow. The esterase can also be used for quantitative evaluation of gene expression using promoter fusions. Substrate containing tricarballylate esters which upon hydrolysis produce a measurable reaction such as but not limited to a color change or fluoresce can be used to measure gene expression. Thus, the coding sequence that codes for the enzyme of this invention can itself be used as a selectable marker, or as a scorable marker by measuring formation of the amino alcohol metabolite or other metabolite.
Another embodiment of the present invention is directed to a DNA construct comprising an expression cassette comprised of:
a) a DNA coding sequence for a polypeptide capable of degrading fumonisin; and
b) control sequences that are operably linked to the coding sequence whereby the coding sequence can be transcribed and translated in a host cell, and at least one of the DNA coding sequences or control sequences is heterologous to the host cell.
Preferred embodiments of the subject invention include a host cell stably transformed by a DNA construct as described above; and a method of producing a polypeptide of a recombinant gene comprising:
a) providing a population of these host cells; and
b) growing the population of cells under conditions whereby the polypeptide encoded by the coding sequence of the expression cassette is expressed;
c) isolating the resulting polypeptide.
A number of expression systems using the said host cells could be used, such as but not limited to, E. coli, yeast or baculovirus. Alternatively, the fumonisin degrading enzymes can be isolated and purified from the seeds or plant parts of a plant expressing the said enzyme.
In yet another embodiment, the present invention is directed to a transgenic plant or plant cells, capable of degrading fumonisin. In another embodiment, the transgenic plant is a maize plant or plant cells capable of degrading fumonisin.
Another embodiment of the subject invention comprises a method of conferring fumonisin degrading abilities to a plant substantially without such abilities comprising transferring to the plant an expressible gene encoding a polypeptide capable of degrading fumonisin.
Additionally, the present invention relates to ruminal microorganisms that have been genetically engineered with the genes imparting fumonisin resistance. These engineered ruminal microorganisms can then be added to feed for consumption by animals susceptible to fumonisin and structurally related mycotoxins.